Identification of genes involved in salinity tolerance in a collection of alfalfa insertional mutants

FERNÁNDEZ, KARINA BEATRIZ; GÓMEZ, CRISTINA; AYUB, NICOLÁS; SOTO, GABRIELA; JOZEFKOWICZ, CINTIA

Rosario, Santa Fé

Congreso; XXXIV Reunión Anual de la Sociedad Argentina de Fisiología Vegetal; 2023

Sociedad Argentina de Fisiología Vegetal

The aim of this work is to identify and characterize new genes associated with salinity tolerance using as a tool a collection of alfalfa (Medicago sativa) mutants that was previously generated in our group through the insertion of the Tnt1 tobacco retrotransposon.To carry out this objective we generated a large collection of mutant lines of Medicago sativa (alfalfa) by activation and transposition of the Tnt1 retrotransposon, from eleven primary transgenic lines (T0) that carry the Tnt1 retrotransposon in alfalfa clones that have good in vitro behavior. By passage through in vitro culture, Tnt1 retrotransposon activates and transposes to new random sites in the alfalfa genome. From eight plants that were molecularly characterized (R1 plants), we generated 153 independent lines (R2 plants) representing an estimated 16000 insertion events in the alfalfa genome, after a second passage through tissue culture. We know insertions are inherited and stable during vegetative growing of alfalfa and in subsequent generations. We crossed Tnt1 mutant lines with elite varieties of alfalfa and performed salinity tolerance assays, during the germination stage and during the implantation stage in hydroponic culture. So far, we selected three mutant lines for their ability to germinate in elevated concentrations of NaCl. Subsequently, we will determine the number of Tnt1 insertions in these lines which show a differential phenotype of salinity tolerance and we will identify the genes that have been repressed or over-expressed by Tnt1 insertion by Inverse PCR (Jozefkowicz et al., 2021).In biotechnological terms this platform represents a powerful tool for the direct identification of new dominant mutations associated with desirable agronomic traits that can be phenocopied using transgenic-free edited cultivars via the CRISPR/Cas9 system in major crops with complex genetics, like alfalfa.Fuentes de financiamiento: PICT 2021 #0466, PD I086 y PD I084 del INTA.Citas: Jozefkowicz, C., Gómez, C., Odorizzi, A., Iantcheva, A., Ratet, P., Ayub N., Soto G. Expanding the Benefits of Tnt1 for the Identification of Dominant Mutations in Polyploid Crops: A Single Allelic Mutation in the MsNAC39 Gene Produces Multifoliated Alfalfa; Frontiers Media. Frontiers in Plant Science; 12; 805032; 12-2021; 1-6.